The goal of this project is to define the role of the membrane environment in two central aspects of T cell receptor (TCR) biology, the assembly of the TCR-CD3 complex and the regulation of receptor phosphorylation. The structures of the extracellular domains of the TCR heterodimer and the CD3 dimers are known, but there is only limited structural information on the transmembrane (TM) and cytoplasmic domains, despite their central role in TCR assembly and signaling. We showed that the assembly of the TCR with the three signaling subunits occurs in the membrane through interaction among a total of nine ionizable TM residues. Each of the three assembly steps involves an interaction between a basic TCR TM residue and a pair of acidic TM residues of the respective signaling module. This assembly mechanism is also relevant for a large number of other activating receptors in the immune system and in each case it involves an interaction between one basic and two acidic TM residues. The NMR structure of the zeta-zeta TM dimer demonstrated that the two aspartic acid residues form a single structural unit at the dimer interface. Also, there is experimental evidence for structural water molecule(s) within the vicinity of the aspartic acid pair which may stabilize this unusual arrangement. During the next funding period, we will determine the NMR structure of a second TM dimer with a TM aspartic acid pair and examine if the placement of the two aspartic acids at the dimer interface and the presence of structural water molecule(s) are common features of such signaling modules. New data also demonstrate that an assembled TM trimer can be expressed in sufficient quantities, and the structure of this TM trimer will be determined in order to define how the basic TM residue of a receptor interacts with the TM aspartic acid pair of a signaling dimer (Aim 1). Preliminary biochemical and NMR studies demonstrate lipid binding of the cytoplasmic domain of CD3 epsilon (Aim 2). Contrary to a previous model which had suggested that ITAMs may peripherally associate with the membrane by forming an extended alpha helix, the NMR data demonstrate that several key residues of the ITAM are actually inserted into the hydrophobic core of the lipid bilayer. This means that the ITAM has to dissociate from the membrane before it can be phosphorylated by Lck. The absence of an alpha-helical periodicity of these protein-lipid interactions further suggests that the actual conformation of the ITAM is distinct from the previously proposed model. This ITAM-lipid interaction is physiologically relevant because new data with a novel FRET-based assay demonstrate binding of the CD3 epsilon cytoplasmic domain to the inner leaflet of the plasma membrane in live cells. This technique enables real- time imaging of one of the earliest events in T cell activation in live cells and will be used to study how the interaction of the ITAM with the membrane is regulated during early stages of T cell signaling. PUBLIC HEALTH RELEVANCE: The plasma membrane provides unique environments for protein-protein and protein- lipid interactions. The major goal of this project is to define how the membrane environment directs the assembly of the TCR-CD3 complex and the initiation of TCR signaling. These issues are likely to be relevant for many other receptors, and the studies described here may thus have a broad impact on our understanding of receptor function.